CTGF has recently emerged as an important growth factor in osteogenesis. Previous studies in our laboratory and others have demonstrated that recombinant CTGF promotes proliferation, matrix production and differentiation of osteoblasts in culture. The importance of CTGF in skeletogenesis was confirmed in CTGF null (KO) mice; they exhibited multiple skeletal dysmorphisms as a result of defects in endochondral ossification. During the previous funding period, we established a colony of heterozygous breeders for the CTGF null (KO) allele (CTGFLacZ), and also generated several lines of transgenic (TG) mice (CTGF pOBCol3.6) that over-express CTGF in bone. More recently, we obtained CTGFflox/+ mice and are in the process of breeding them with Col I 3.6-Cre mice to generate osteoblast-specific CTGF null mice. Preliminary data from KO and TG mice support the hypothesis that CTGF is an anabolic bone growth factor that directly regulates osteoblast differentiation and function. In addition to its effects on osteoblasts, we present data showing that CTGF ablation (KO mice) or over-expression (TG mice) affects osteoclast formation. We hypothesize that these abnormalities in osteoclast number are secondary to altered production of osteoclastogenic factors (e.g. RANK-L) by stromal cells/osteoblasts in the bone microenvironment. Having these genetically-engineered mice in hand, studies in Aim 1 propose to evaluate the effects of CTGF absence (KO) or over-expression (TG) on bone in vivo, and to assess the differentiation and function of bone cells (osteoblasts and osteoclasts) derived from these mice in primary cultures. Studies have established that CTGF can function as an ECM-associated (matricellular) protein through unique interaction with specific integrins expressed by a given target cell, and the subsequent activation of integrin signaling pathways provides a mechanistic interpretation for some of its biological activities. We have demonstrated that osteoblasts attach to CTGF via the av5 integrin, resulting in the formation of focal adhesions, cytoskeletal reorganization and the activation of FAK and Erk. We also have preliminary data demonstrating that osteoclasts attach to CTGF via the av3 integrin. Studies in aim 2 will test the hypothesis that CTGF acts as a matricellular protein that binds to specific cell surface integrins on osteoblasts and osteoclasts to initiate integrin-activated signaling, cytoskeletal reorganization, and regulate cell function. We recently demonstrated that TGF-1 is a potent inducer of CTGF expression and that CTGF is a downstream mediator of TGF-1-induced ECM production in osteoblasts. We present preliminary data that Smad, Erk and Src signaling are required for TGF-1 up-regulation of CTGF in osteoblasts, and studies in Aim 3 propose to investigate the interaction between Src and Erk, the effects of activating Smad, Src and/or Erk signaling on CTGF expression in the absence of TGF-1, the mechanism whereby Erk potentiates Smad signaling, and the requirement for Src, Erk and Smad signaling for CTGF dependent ECM production in osteoblasts. Proposed experiments are expected to generate novel information regarding the effects of CTGF deficiency or over-expression on bone development in vivo, its mechanisms of action on osteoblasts and osteoclasts, and the molecular requirements for CTGF induction by TGF-1 in osteoblasts. PUBLIC HEALTH RELEVANCE. Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. CTGF is a novel growth factor in bone and the proposed studies will generate new information regarding its effects and mechanisms of action on bone cell development and function. Once we understand its full effects on bone and how it works to promote bone formation, this information will be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with clinically significant bone loss.